The object of the invention is a novel process for sensitizing antigen-presenting cells, novel means for the implementation of the process and novel membrane vesicles having an immunogenic potency.
Since the demonstration of the existence of CD8+ cytotoxic T lymphocytes specific for tumor antigens presented in the context of class I molecules (Rosenberg et al., 1996; Boon, 1992), several laboratories have been able to show that anti-tumor immuno-therapy is an efficacious therapeutic strategy in animal models (Pardoll, 1995). The principle of immunotherapy is to induce an effective immune response against specific tumor antigens. Up to the present, it has been possible to carry this out in different ways. First, tumor cells expressing recombinant co-stimulatory molecules and/or immunomodulatory cytokines are capable of stimulating anti-tumor responses capable of eradicating solid tumors in vivo (Zitvogel et al., 1996 {a}). Similarly, peptide derivatives of tumor antigens (or exogenous antigens expressed in tumor cells) injected in different chemical forms including the use of liposomes or viruses (adenovirus or poxvirus, for example) as vectors are capable of causing tumors to regress. Finally, professional antigen-presenting cells, such as dendritic cells sensitized with peptides derived from tumor antigens, reinjected in vivo induce potent anti-tumor responses as well as the regression of solid tumors established in mice (Mayordomo et al., 1995).
Immunotherapy based on the use of dendritic cells has been able to show its efficacy in the studies conducted in the mouse. As a result, this therapy has recently been transposed to the clinic. In the Unites States, trials are presently underway to demonstrate that dendritic cells loaded with tumor peptides significantly increase the frequency of specific cytotoxic T lymphocytes (CTL).
A first limitation to this approach is the sensitization of the dendritic cells with peptides derived from tumor antigens. In fact, in the majority of tumors specific antigens have not been identified. Antigens specific to the tumors are only known in cases of tumors induced by viruses (carcinoma of the uterine cervix), in cases of melanoma (self antigens, mutated antigens, differentiation antigens) or in a small percentage of breast tumors (oncogenes or products of tumor suppressor genes having undergone mutations). However, the direct implication of these peptides or tumor antigens in the elimination of the tumors in man remains to be demonstrated. Novel sensitization methods of the antigen-presenting cells such as dendritic cells thus prove to be necessary. The object of these methods is to induce specific anti-tumor responses in the context of class I and class II molecules of the MHC.
Most of the sensitization methods of dendritic cells at present use peptides corresponding to epitopes presented in combination with the class I molecules and identified in tumor cells by means of CTL clones specific for the tumor. However, these methods are probably not optimal since they do not take into account epitopes recognized in the context of the class II molecules which are critical for the proliferation of the helper T lymphocytes necessary for obtaining optimal cytotoxic responses. Furthermore, epitopes presented by the tumor cells and those presented by antigen-presenting cells (as for example the dendritic cells) are probably not the same. Finally, tumor peptides recognized by the CTL are only available in the case of a small percentage of patients having molecules of the appropriate class I haplotype.
The ideal sensitization method, one which would be applicable to any tumor with a minimal risk of immunoselection, must not be limited to a small number of identified tumor antigens. Similarly, such a method ought to make use of intact protein antigens rather than peptides in order to enable the dendritic cell to prepare them and to present the adequate combination of peptides in combination with the class I and class II molecules, and do so for any individual.
Recently, Gilboa and collaborators (Boczkowsky et al., 1996) have been able to show that messenger RNAs prepared from tumors biopsies loaded in the dendritic cells may have an in vivo anti-tumor effect. However, the RNAs are very unstable and the quantity of potentially interesting RNA compared with the total RNA is probably very low. Zitvogel et al. (Zitvogel et al., 1996 {b}) have shown that tumor peptides prepared from an acidic tumor eluate (acidic peptide eluate: APE) may be used to load dendritic cells. These cells thus loaded, once injected, have the capacity to cause tumors to regress. However, in the case of tumors which do not express tumors of class I (which represent the majority of metastatic human tumors) or in the case of tumors which may not be dissociated in a cellular suspension, the approach using the acidic eluates is not very efficacious and not reproducible.
A second limitation to immunotherapy based on the use of dendritic cells is linked to the phenotypic changes which may occur when these cells are maintained in culture or subjected to different treatments. This may in fact lead to cell populations which are not very homogeneous and inadequately characterized for therapeutic use.
Hence there exists a real need to improve the methods for sensitizing antigen-presenting cells in order to enhance the efficacy of these approaches and to broaden their applications as well as to develop novel means for the vectorization of antigens and other molecules.
The present invention provides solutions to these questions. The object of the present invention is in fact to provide novel methods for sensitizing antigen-presenting cells, in particular dendritic cell, as well as for the identification, isolation and characterization of the novel membrane vesicles having remarkable immunogenic properties.
One of the features of the invention is more particularly to provide a novel reproducible process for sensitizing antigen-presenting cells by tumor antigens.
Another feature of the invention is to provide a novel reproducible process for sensitizing antigen-presenting cells by tumor antigens, in which it is not necessary that the tumor antigens are known.
Another feature of the invention is to provide the means which make it possible to set up a library of tumor antigens.
Another feature of the invention resides in lipid membrane vesicles produced by the tumor cells or by the dendritic cells and endowed with immunogenic properties as well as their use for the production of antigen libraries, the sensitization of antigen-presenting cells or the vectorization of antigens, in particular in the context of immunotherapeutic approaches.
In this respect a first object of the invention relates to a vesicle derived from tumor cells having the following characteristics:
it is freed from its natural environment,
it comprises a lipid bilayer (designated by xe2x80x9csurfacexe2x80x9d) which surrounds a cytosolic fraction,
and optionally,
it exhibits on its surface molecules of class I of the major histocompatibility complex (MHC) and/or of class II of the major histocompatibility complex (MHC), optionally loaded with antigenic peptides and/or adhesion molecules and/or lymphocytic costimulatory molecules, and/or,
it contains in its cytosolic fraction tumor antigen molecules and/or immunomodulators and/or chemo-attractors and/or hormones and/or nudeic acids.
The secretion of the vesicles by cells is a phenomenon described in the prior art (reticulocytes, B lymphocytes, macrophages). These vesicles are usually designated by the generic term xe2x80x9cexosomexe2x80x9d which reflects their mechanism of production by exocytosis of internal vesicles. However, the physiological role of these vesicles has not been really established. Furthermore, the structural characteristics, properties and functions of these vesicles vary depending on the cell type from which they are derived.
Unexpectedly, the inventors have now demonstrated that tumor cells are capable of secreting vesicles exhibiting particularly interesting immunogenic properties. These vesicles usually correspond to an internal vesicle contained in an endosome of a tumor cell and secreted by said tumor cell subsequent to the fusion of the external membrane of said endosome with cytoplasmic membrane of above-mentioned tumor cell. Owing to this mechanism of formation, their cellular origin and their original functional properties and characteristics, these vesicles are designated in what follows by the term  less than  less than texosome  greater than  greater than .
The expression xe2x80x9cfreed from its natural environmentxe2x80x9d signifies that the vesicle is separated physically from the cell from which it is derived or even that it is partially isolated or purified. Usually, the vesicle is thus produced by the cell by means of exocytosis, then partially isolated and purified so as to produce an enriched composition. This expression may also signify that not only the vesicle was secreted by the cell at the moment of fusion of the multivesicular endosomes with the plasma membrane, but that it is no longer surrounded by soluble elements which are in the lumen of the endosome, or that it lacks intact cells. The expression xe2x80x9cderived from a tumor cellxe2x80x9d signifies that the vesicle possesses structural elements of a tumor cell. This vesicle is usually xe2x80x9cderivedxe2x80x9d from a tumor cell in the sense in that it is produced, at least in part, then released by a tumor cell, at a given stage of its development.
According to an advantageous embodiment of the invention, the texosomes of the invention exhibit MHC molecules loaded with antigenic peptides and/or express adhesion molecules and/or express lymphocytic costimulatory molecules, but lack in their cytosolic fraction tumor antigenic molecules and immuno-modulators and nucleic acids.
According to another advantageous embodiment the texosomes of the invention are such that the molecules of the MHC are xe2x80x9cemptyxe2x80x9d, i.e. not loaded with antigenic peptides and the texosomes comprise in their cytosolic fraction tumor antigenic molecules, immunomodulators and/or nucleic acids. Texosomes having empty MHC molecules may be obtained either from tumor cells exhibiting for example a deficiency of the peptide transporter (TAP) or by washing of texosomes or tumor cells in order to eluate the peptides associated with the molecules of the MHC.
According to an advantageous embodiment of the invention, the texosomes of the invention are such that the molecules of the MHC are loaded with antigenic peptides and/or express adhesion molecules and/or lymphocytic costimulatory molecules and the texosomes contain in their cytosolic fraction tumor antigenic molecules, immunomodulators and/or nudeic acids.
The term xe2x80x9ctumor cellsxe2x80x9d embraces in a general manner any cell derived from a tumor, for example a solid or liquid tumor, as well as the cells transformed or immortalized in vitro. Preferably, it refers to a solid, ascitic or hematopoietic tumor.
As examples, mention may be made of the cancer cells of the malignant melanoma type (derived from primary lines established xe2x80x9cex vivoxe2x80x9d or even dissociated cells derived from the operating theatre) which express at their surface peptides like MART-1/Melan-A in the context of MHC class 1, HLA-A 02-01, and containing the protein antigen MART-1.
Mention may also be made of cells derived from renal cancer (clear cell adenocarcinoma) or leukemias the cells of which express specific translocation products.
Thus, the antigenic peptides likely to load the molecules of the MHC are derived from the following antigens for example: those derived from melanomas such as: MART-1, tyrosinase, MAGE-1/2/3, P53 (in different tumors) or HER2/Neu, PSA, CEA or also PSMA. Other tumor antigens are cited for example in the article by Rosenberg (Immunology Today 18 (1997) 175) incorporated into the present description as a reference.
More generally, mention may be made of fusion/translocation products, products of oncogenes or anti-oncogenes, or even differentiation antigens or peptides of self or mutated peptides.
By lymphocytic costimulatory molecules is meant for example molecules which give to the T lymphocytes additional signals to those given on interaction of the complexes molecule of class I and class IIxe2x80x94peptide with the T cell receptor.
As examples, mention may be made of:
CD80, CD86, ICAM, LFA, CD40, certain members of the TNF R family and adhesion or chemo-attraction molecules (permitting contact between the professional antigen-presenting cell and the effector lymphocytes, or the intracellular transport/specific localization (xe2x80x9ctrafficking/homingxe2x80x9d) of other cells to the vaccinal or inflammatory site.
The tumor antigenic molecules contained in the cytosol or presented by the texosomes derive from proteins expressed selectively and/or abundantly by the tumor cells.
The immunomodulators which may be present in the cytosol of the texosomes are, for example
TNF-xcex1, or
interleukin 1, or
interleukin 15, or
C-CR (chemokines).
The nudeic acids likely to be present in the cytosol of the texosomes are derived from the tumor cell itself. These nucleic acids are found in the cytosol of the texosomes as a direct consequence of their mechanism of formation. They may also be heterologous nucleic acids.
More special characteristics of the texosomes of the invention are the following:
they are small membrane vesicles of about 60 to 100 nm, most often about 60 to 90 nm, in particular 60 to 80 nm, secreted by the tumor cells,
they possess molecules usually present in the endosomes,
they contain tumor antigens, like for example MART-1 in the case of melanoma cells,
they lack dead cells and/or cellular debris
they lack contaminants such as membrane contaminants, endoplasmic reticulum, Golgi apparatus, mitochondria or nuclear constituents,
they bear at their membrane functional molecules of class I/II loaded with tumor antigenic peptides,
they can stimulate the proliferation of specific T lymphocytes in vitro
they can sensitize in vivo and in vitro dendritic cells which are then capable of activating the tumor-specific T cells,
they possess the capacity when they are inoculated in vivo in particular intradermally, to cause established solid tumors to regress,
they bear lymphocytic costimulatory molecules such as CD40 and CD80, and/or,
they contain the (xe2x80x9cheat-shockxe2x80x9d) protein HSP70,
they lack the protein gp96,
they contain interleukins or chemo-attractants or immunomodulators.
Another interesting characteristic of the texosomes is that they contain phosphatidylserine in the external layer. Phosphatidylserine (PS) is one of the major components of cell membranes, usually present very largely in the internal layer of the lipid bilayer. Under certain circumstances, such as the early steps of apoptosis, the PS is redistributed towards the external layer. The presence of PS in the external layer of the cytoplasmic membrane of the apoptotic cells constitutes a signal for recognition by the macrophages. In order to determine whether the PS is exposed at the surface of the texosomes, preparations of exosomes purified from supernatants of FON human melanoma cells were analyzed by the method described by Aupeix et al. (J. Clin. Invest. 99: 1546-1554, 1997). The phosphatidylserine content in the external layer of the FON samples (containing 390 microg/ml of proteins) is 460 nM of PS. The exosomes thus contain considerable quantities of PS in their external layer.
Assays making it possible to verify that the texosomes of the invention possess molecules usually present in the endosomes consist of electron microscopy and immunoblotting (Western blot). These assays make it possible to show that the texosomes of the invention express the transferrin receptor for transferrin, LAMP (xe2x80x9clysozyme associated membrane proteinxe2x80x9d) molecules, molecules of class I/II, tumor antigens.
An assay making it possible to verify that the texosomes of the invention lack contaminants is electron microscopy and immuno-blotting with antibodies to calnexin which is present in the endoplasmic reticulum.
An assay making it possible to verify that the texosomes bear at their membrane functional molecules of class I/II loaded with tumor antigenic peptides consists of an antigenic presentation to T lymphocytes specific for the antigens of the tumor concerned (proliferation tests of T clones specific for antigens and restricted class I MHC).
It is also possible to use a test of secretion of cytokines (IFNxcex3, GM-CSF, TNFxcex2) by the above-mentioned T clones.
An assay making it possible to verify that there is in vivo and in vitro sensitization of the dendritic cells capable of activating tumor-specific T cells is given in FIG. 7 (proliferation and/or secretion test of cytokines by antigen-specific T clones by the xe2x80x9ccross-primingxe2x80x9d method: texosomes of a tumor MART-1+, HLA-A2-loaded one to a dendritic cell MART-1-, HLA-A2+).
An assay making it possible to verify that the texosomes possess the capacity when they are inoculated, in particular intradermally, to cause established solid tumors to regress is given in FIG. 6.
As an example, 10 to 40 xcexcg of texosomes of tumor are injected intradermally on the same side as the tumor established 3 to 10 days previously; the animal bearing the tumor and the progressive disappearance of the tumor established 7 to 10 days previously are observed (in rodents, such as mice).
An advantageous texosome of the invention is constituted by a texosome such as that defined above and exhibiting on its surface class I and/or class II molecules of the MHC, optionally loaded with antigenic peptides and containing in its cytosolic fraction tumor antigenic molecules. More particularly, the preferred texosome also comprises one or more lymphocytic costimulatory molecules and/or the protein HSP70. In a particular embodiment, the texosome lacks the protein gp96.
According to an advantageous embodiment, the invention relates to a texosome such as that defined above,
expressing on its surface class I and/or class II molecules of the major histocompatibility complex (MHC), and/or antigens characteristic of tumors and/or lymphocytic costimulatory/adhesion molecules and/or immunomodulators and/or chemo-attractants, exogenous with respect to the tumor cell from which the exosome is derived, or
containing tumor antigens and/or immunomodulators and/or nucleic acids or cytotoxic agents or hormones exogenous with respect to the tumor cell from which the exosome is derived.
The invention also relates to a process for the preparation of texosomes such as those defined above. This process advantageously comprises a step in which a biological sample is provided and a step involving the isolation of texosomes from said sample.
The biological sample is advantageously constituted of membrane fractions, culture supernatants or lysates of tumor cells or even fresh tumor suspensions.
The biological sample may be tumor fragments obtained from operations after surgical excision (1st case) or even from organs bearing tumors (surgically excised organ) (2nd case) which are treated by mechanical dissociation (1st case) or by prolonged perfusion (2nd case).
The final cellular suspension is treated in the same manner as the culture supernatants.
The sample may also be cells treated by several successive cycles of freezing/thawing.
According to an advantageous embodiment, the biological sample used in the process of the invention is:
a blood sample taken from the efferent vein of the isolated tumor-bearing organ, or
a plasma or serum sample of the circulating blood of a patient, or
the drainage product (physiological serum possibly containing dexamethasone or a cytotoxic agent stimulating exocytosis of the texosomes) of an organ excised surgically and treated ex vivo by isolated perfused circuit for the drainage of the tumor it bears or also
the supernatant of a tumor explant dissociated in vitro.
The efferent blood sample of the isolated tumor-bearing organ corresponds to 20 to 50 ml of blood taken from the principal efferent vein of the tumor-bearing organ, taken prior to surgical ablation.
The drainage product of an organ excised surgically and treated ex vivo by isolated-perfused circuit is obtained in the following manner.
In the case of an organ presenting an afferent artery and an efferent vein, the artery is characterized by a small plastic tube connected to an up- inclined pouch containing physiological serum with optionally other agents. The organ is drained and the liquid is returned by another small tube catheterizing the down- inclined vein (for example in the case of renal cancer or a cerebral glioblastoma).
The object of dexamethasone, optionally contained in the drainage product, is to increase the cellular stress and the exocytosis of the texosomes from the tumor cell.
The supernatant of a tumor explant dissociated in vitro is obtained in the following manner:
the mechanical dissociation of the tumor is performed leading to a unicellular suspension containing tumor cells and tumor stroma cells and cells of the immune system; this suspension may be irradiated and recovered for differential centrifugations.
As indicated above, in a particular embodiment of the invention the biological sample may be treated by one or more agents stimulating the production of texosomes. This treatment may comprise the addition of steroid agents (dexamethasone, for example), pharmacological agents (for example cytotoxic agents such as taxanes, cis-platinum, etc), agents likely to increase the quantity of multivesicular endosomes and/or irradiation of the sample.
As regards the irradiation, it must be sufficient to induce the cytostatic action of the tumor cells. The irradiation of the tumor cells may be done prior to placing them in culture or during or after placing the tumor cells in culture. Moreover, it is advisable to irradiate when the tumor cells are alive, i.e.:
either on the excised organ bearing the tumor prior to perfusion,
or on the cells in culture,
or on the mechanically dissociated cell suspension; but, in all cases, prior to tumor cell stress due to hypoxia/vascular necrosis/dehydration.
As regards the treatment with steroids, it enables cellular activation to be induced, leading to exocytosis of the texosomes.
As regards the treatment with pharmacological agents,it makes it possible:
to modify the cytoskeleton and rearrange the intracellular compartments in order to perturb the phenomena of internalization and exocytosis,
to depolymerize the microtubules.
As regards the treatment with an agent likely to increase the quantity of multivesicular endosomes, it is performed during the placing of the cells in culture; as agent mention may be made of nocodazole (drug leading to the depolymerization of the microtubules), bafilomycin (drug inhibiting the vacuolar ATPases) (xe2x80x9cBafilomycins: A class of inhibitors of membrane ATPases from microorganisms, animal cells and plant cellsxe2x80x9d (1988) Proc. Natl. Acad. Sci. USA 85: 7972-7976).
An advantageous process for the preparation of texosomes according to the invention is carried out:
a) either on cultures of tumor cells, and comprises:
irradiation of the tumor cells before, during or after placing them in culture, at an intensity sufficient to induce the cytostatic action of the tumor cells and not exceeding 15,000 rads, and advantageously about 10,000 rads, or
a treatment during culture of the tumor cells with steroids, for example dexamethasone or cytotoxic agents, for example 5-fluorouracil (5-FU) or cis-platinum, docetaxel, anthracyclin, a spindle poison, antipyrimidine or interleukin for example IL10, IL2, IL15, GM-CSF, or,
a treatment with an agent capable of increasing the quantity of multivesicular endosomes, for example nocodazole (Gruenberg J et al., (1989) xe2x80x9cCharacterization of the Early Endosome and Putative Endocytic Carrier Vesicles in vivo and with an Assay of Vesicle Fusion in vitroxe2x80x9d The Journal of Cell Biology 108: 1301-1316) and hence increase the production of texosomes,
b) or on a sample of the physiological serum draining a surgically excised organ and treated ex vivo by isolated-perfused circuit for the draining of the tumor which it bears, or
c) or on the supernatant of a tumor explant dissociated in vitro and comprising:
a treatment with steroids, for example dexamethasone, or cytotoxic agents, for example 5-fluorouracil (5-FU); cis-platinum, taxanes or an interleukin for example IL-10, IL-2, GM-CSF.
The texosome isolation step may be performed according to different processes such as centrifugation, chromatography, electrophoresis, nanofiltration, etc. For example, it may involve a differential centrifugation of membrane fractions of culture supernatants or lysates of tumor cells or fresh tumor suspensions and recovery of the fraction(s) containing said exosomes (Raposo et al., J. Exp. Med. 1996, 183: 1161-1172). In this particular embodiment, the supernatant membrane fraction is that obtained after centrifugation at 100,000 g. It may advantageously involve a liquid phase electrophoresis which leads to the separation of biological materials according to their charge. The examples which follow show that this process may be advantageously used for the isolation of texosomes in good yields. This process is moreover particularly advantageous on an industrial scale.
The object of the invention is also a preparation process for texosomes such as defined above, comprising in addition:
either the genetic modification of the tumor cells by exogenous genes coding for class I and/or class II molecules of the major histocompatibility complex (MHC), and/or genes coding for antigens characteristic of tumors and/or genes coding for costimulatory/adhesion molecules or attractant chemokines, since it is possible to find the products of these exogenous genes expressed at the surface of the texosomes and/or be sequestered within the interior of the texosomes,
or the in vitro modification of the texosomes produced by tumor cells, such as the introduction (by electroporation, by fusion with a synthetic liposome, by recombinant virus or by a chemical method) of proteins or nucleic acids or pharmaceutically defined medicines in and/or with the texosomes.
The invention also relates to texosomes capable of being obtained according to the process described above.
The texosomes of the tumor cells transfected as indicated above are recovered and used as tumor vaccines.
The texosomes modified in vitro as indicated above are designed to deliver the exogenous material to a target cell in vitro or in vivo.
As regards the fusion with a synthetic liposome, this process is performed for example as indicated in Nabel et al. (1996) or in Walker et al. (1997, Nature 387 pages 61 et seq.).
The invention also relates to antigen-presenting cells, in particular to B lymphocytes, macrophages, monocytes or dendritic cells, loaded with texosomes as defined above. Advantageously, they are dendritic cells.
The dendritic cells of the invention have, in particular, the following characteristics:
in tumor models which do not express class I molecules and which, consequently, do not have the capacity to stimulate CD8+ T cells, dendritic cells loaded with texosomes of tumor cells may present these tumor peptides to cytotoxic T cells in the context of the class I molecules of the MHC (characteristic No. 1)
dendritic cells loaded with texosomes of tumor cells injected intravenously or subcutaneously are also very efficacious (characteristic No. 2).
The following assay enables the characteristic No. 1 to be demonstrated.
In the human system, a class I-negative texosome, incubated in the presence of a class I-positive dendritic cell may lead to the stimulation of CD+8 T clones specific for the antigen contained in the texosome (see FIG. 7).
The following test enables the characteristic No.2 to be demonstrated.
In a mouse system in which the tumor is class I-negative and in which the texosomes themselves also lack class I molecules, these texosomes may, when they are incubated and loaded on to the dendritic cells, mediate an anti-tumor immune response whereas alone after intradermal injection they cannot.
The invention also relates to a preparation process for antigen-presenting cells such as defined above, comprising the steps of incubation of the antigen-presenting cells in the presence of texosomes such as defined above and recovery of above-mentioned antigen-presenting cells loaded with above-mentioned texosome.
The invention also relates to presenting cells loaded with texosomes and capable of being obtained by the process described above.
The object of the invention is also the use of texosomes such as those defined above for the sensitization of antigen-presenting cells, in particular B lymphocytes, macrophages, monocytes or dendritic cells or for the stimulation of specific T lymphocytes.
The invention also relates to a membrane vesicle freed from its natural environment, secreted by antigen-presenting cells loaded with texosomes such as defined above.
In order to obtain these membrane vesicles defined above, recourse may be had to a process comprising:
a step involving the preparation of a texosome such as that defined above,
a step involving the incubation of a texosome with antigen-presenting cells,
a step involving the differential centrifugation of membrane fractions of culture supernatants or lysates of the above-mentioned antigen-presenting cells, loaded with texosomes and
a step involving the recovery of the fraction containing the above-mentioned membrane vesicles.
The object of the invention is also membrane vesicles such as defined above and capable of being obtained according to the process described above.
In this respect the invention relates to membrane vesicles produced by dendritic cells. Unexpectedly, the inventors have in fact demonstrated that the dendritic cells were capable of producing membrane vesicles having particularly advantageous immunogenic properties. Such vesicles have in particular been visualized, isolated and characterized from culture supernatants of dendritic cells, in particular immature human dendritic cells. Unlike vesicles described up to now, these vesicles are very advantageous to the extent that they present simultaneously and in considerable numbers molecules of classes I and II of the major histo-compatibility complex. These membrane vesicles comprise a lipid bilayer surrounding a cytosolic fraction and are designated in what follows by the term xe2x80x9cdexosomexe2x80x9d on account of their origin and their unusual biological and biochemical properties. These vesicles indeed possess remarkable immunogenic properties because they are capable of stimulating the production and the activity of cytotoxic T lymphocytes both in vivo and in vitro, and because they enable the growth of established tumors to be suppressed in vivo, in a manner dependent on MHC-restricted T lymphocytes. The dexosomes hence constitute active principles particularly suited to non-cellular approaches to immunotherapy.
Special membrane vesicles in the sense of the invention are hence vesicles capable of being produced by dendritic cells, and which bear one or more class I molecules of the major histocompatibility complex and one or more class II molecules of the major histocompatibility complex.
The dexosomes advantageously bear lymphocytic costimulatory molecules, and in particular molecules CD63 and/or CD82 and/or CD86, and preferably at least CD86. The studies presented in the examples show in fact that the dexosomes are strongly labelled by antibodies directed specifically against these costimulatory molecules.
Moreover, the electron microscopical analyses show that the dexosomes are homogeneous and possess a diameter included between about 60 and 100 nm, most frequently between about 60 and 90 nm.
A particularly preferred variant of the invention is thus represented by a dexosome having a diameter included between about 60 and 90 nm, obtained from a dendritic cell, and comprising:
one or more class I molecules of the major histocompatibility complex,
one or more class II molecules of the major histo-compatibility complex,
one or more CD63 molecules,
one or more CD86 molecules; and
one or more CD82 molecules.
In a particular embodiment of the invention, the dexosomes comprise in addition one or more antigenic peptides and/or are obtained from immature dendritic cells.
Still in accordance with a particular embodiment, the dexosomes lack H2-M markers, the li chain and calnexin (a specific marker of the endoplasmic reticulum).
Moreover, still in accordance with an advantageous embodiment, the dexosomes of the invention contain in addition phosphatidylserine (PS) in their external layer. Thus, exosome preparations purified from dendritic cell supernatants derived from bone marrow were analyzed by the method described by Aupeix et al. (J. Clin Invest. 99: 1546-1554, 1997). The phosphatidylserine content in the external layer of the BMDC samples (containing 35 microg/ml of proteins) is 80 nM of PS. The dexosomes thus contain considerable quantities of PS in their external layer.
The dexosomes can be prepared according to a methodology comprising a first step for obtaining dendritic cells or a cell culture containing dendritic cells, an optional second step during which the cells may be sensitized to antigens of interest, and a third step comprising the production of dexosomes from these cell cultures. These different steps may be advantageously carried out according to the methodologies described hereafter.
The first step of the process comprises the the provision of (a) culture(s) of dendritic cells. They may be cultures of cells enriched in dendritic cells, even cell cultures consisting essentially of dendritic cells. Advantageously, they are obviously human dendritic cells.
The preparation of dendritic cells has been well documented in the literature. Thus, it is known that these cells can be obtained from stem cells of the immune system or from monocyte precursors or even isolated directly in a differentiated form (review by Hart, Blood 90 (1997) 3245).
The production of dendritic cells from stem cells is illustrated for example by Inaba et al. (J. Exp; Med. 176 (1992) 1693), Caux et al. (Nature 360 (1992) 258) or Bernhard et al. (Cancer Res. 55 (1995) 1099). These publications show in particular that dendritic cells can be produced by culture of bone marrow in the presence of granulocyte-macrophagexe2x80x94colony stimulation factor (GM-CSF) or, more exactly, from hematopoietic stem cells (CD34+) by culture in the presence of a combination of cytokines (GM-CSF+TNFxcex1).
The production of dendritic cells from monocyte precursors is illustrated for example by Romani et al. (J. Exp. Med. 180 (1994) 83), Sallusto et al. (J. Exp. Med. 179 (1994) 1109), Inaba et al. (J. Exp. Med. 175 (1992) 1157) or also Jansen et al. (J. Exp. Med. 170 (1989) 577). These methodologies are based essentially on the collection of mononucleated cells in blood and placing them in culture in the presence of various combinations of cytokines. A specific method consists of treating the monocyte precursors of the blood in the presence of combinations of cytokines such as interleukin4+GM-CSF or interleukin-13+GM-CSF for example. This process is also illustrated by Mayordomo et al., 1995. Moreover, it is also possible to treat the monocyte precursors with pharmacological agents for cellular differentiation, such as calcium channel activators.
Another approach to the production of dendritic cells consists of isolating differentiated dendritic cells from biological samples. This approach has been described for example by Hsu et al. (Nature Medicine 2 (1996) 52). The methodology described by that group consists essentially of harvesting peripheral blood samples and of subjecting them to different gradients and centrifugations so as to extract the dendritic cells from them.
The preferred methodology in the framework of the present invention is based on the production of dendritic cells from monocyte precursors or from bone marrow. These methodologies are illustrated in the examples. More particularly, preference is given to the use in the framework of the present invention of dendritic cells obtained by treatment of monocyte precursors (contained in the blood or bone marrow) in the presence of a combination of GM-CSF+IL-4 or GM-CSF+IL-13.
Moreover, for the implementation of the present invention, it is quite especially advantageous to use a population of dendritic cells comprising immature dendritic cells. Advantageously, a population of dendritic cells composed mainly (i.e. to at least 60%, preferably 70%) of immature dendritic cells is used. The immature state of the dendritic cells corresponds to an early stage of their development, at which they exhibit a high endocytic activity and express low levels of classes I and II molecules of the MHC and lymphocytic costimulatory molecules at their surface. Surprisingly, the inventors have indeed found that only immature dendritic cells were capable of producing membrane vesicles in significant quantity. This discovery is all the more surprising as the dendritic cells at the immature stages are known for their low capacity to stimulate the T lymphocytes and hence for their low biological activity (Cella, Nature London, 388 (1997) 782).
The first step of the process of the invention may thus advantageously comprise the preparation of a population of dendritic cells comprising immature dendritic cells, in particular starting from monocyte precursors, more particularly by treatment with a combination of cytokines such as GM-CSF+IL-4 or GM-CSF+IL-13.
Moreover, it is also possible to use in the framework of the present invention immortalized populations of dendritic cells. They may be immortalized lines of dendritic cells (line D1 used in the examples or any other line produced by example by introduction of the myconcogene into the dendritic cells). They may also be dendritic cells prepared and then immortalized in vitro. The value of immortalized dendritic cells resides in the constitution of libraries of cells sensitized to given groups of antigens, which can be used industrially to prepare dexosomes capable of being administered to whole families of patients.
Once the dendritic cells are prepared they may be maintained in culture, purified further, stored or used directly in the following steps of the process.
The dexosomes of the invention can be prepared from dendritic cells not loaded with antigens, i.e. not bearing specific antigens in their membranes or their cytosol. Such dexosomes are then designated as being xe2x80x9cnaivexe2x80x9d or xe2x80x9cvirginxe2x80x9d.
According to a preferred embodiment, the dexosomes of the invention are however prepared from dendritic cells sensitized to an antigen or to a group of antigens. In this embodiment, the dexosomes carry themselves said antigen(s) and are thus capable of inducing a response to them.
Different processs may be used to sensitize the dendritic cells to antigens. These processs have been mentioned above and comprise in particular:
the placing of the dendritic cells in contact with antigenic peptides (xe2x80x9cpeptide pulsingxe2x80x9d). This approach consists of incubating the dendritic cells for a variable time (usually from about 30 minutes to about 5 hours) with one or more antigenic peptides, i.e. with a peptide derived from an antigen, such as might result from the treatment of said antigen with an antigen-presenting cell. This type of approach has been described for example for antigenic peptides of the HIV virus, influenza virus or HPV or for peptides derived from the antigens Mut1, Mart, Her2 or Neu for example (Macatonia et al., J. Exp. Med. 169 (1989) 1255; Takahashi et al., Int. Immunol. 5 (1993) 849; Porgador and Gilboa, J. Exp. Med. 182 (1995) 255; Ossevoort et al., J. Immunother. 18 (1995) 86; Mayordomo et al., previously mentioned; Mehta-Damani et al., J. Immunol (1994) 996). It is also possible to incubate the dendritic cells with an acidic peptide eluate of a tumor cell according to the methodology described by Zitvogel et al. (1996, previously mentioned).
the placing of the dendritic cells in contact with one or more antigens (xe2x80x9cantigen pulsingxe2x80x9d). This approach consists of incubating the dendritic cells not with one or more antigenic peptides but with the intact antigen(s). The value of this process resides in the fact that the antigen will be converted into antigenic peptides by the natural mechanisms of the dendritic cell, so that the resulting antigenic peptides presented by the dendritic cell ought to provide a better immunogenicity. This approach has been illustrated for example by Inaba et al. (J. Exp. Med. 172 (1990) 631) or by Hsu et al. (Nature Medicine 2 (1996) 52).
the placing of the dendritic cells in contact with one or more antigenic protein complexes. This approach is similar to the preceding one but may increase the efficacy of processing and/or presentation of the antigen. In particular, the antigen may be used in a soluble form or complexed with targetting elements which enable, in particular, membrane receptors like the mannose receptors or the immunoglobulin receptors (Rfc) to be targetted. It is also possible to make the antigen particulate so as to improve its penetration or even its phagocytosis by the cells.
the placing of the dendritic cells in contact with cells or membranes of cells expressing antigens or antigenic peptides. This process is based on the direct transfer of antigens or antigenic peptides by fusion of cells or cell membranes. This approach has been illustrated for example by the fusion between dendritic cells and membranes of tumor cells (Zou et al., Cancer Immunol. Immunother. 15 (1992) 1).
the placing of the dendritic cells in contact with membrane vesicles containing antigens or antigenic peptides (in particular exosomes from tumor cells such as already described above). This approach to sensitization of the dendritic cells using exosomes such as demonstrated in the present invention, is particularly advantageous in as much as it does not require knowledge of particular antigens and in as much as the antigen peptides loaded are in a native conformation. This technology is illustrated in the examples.
the placing of the dendritic cells in contact with liposomes containing antigens or antigenic peptides (Nair et al., J. Exp. Med. 175 (1992) 609).
the placing of the dendritic cells in contact with RNAs coding for antigens or antigenic peptides (see Boczkowsky et al., 1996, previously mentioned).
the placing of the dendritic cells in contact with DNAs coding for antigens or antigenic peptides (possibly incorporated in vectors of the plasmid, viral or chemical type). Thus, one method of sensitizing the dendritic cells consists for example of infecting the dendritic cells with a virus against which protection is desired. This has been described for example for the influenza virus (Bhardwaj et al., J. Clin. Invest. 94 (1994) 797; Macatonia et al., previously mentioned). Another approach consists of delivering, by means of a virus or other nucleic acid transfer vectors, a DNA coding for the antigen(s) or antigenic peptides of interest. Such an approach has been illustrated for example by Arthur et al. (Cancer Gene Therapy, 1995) or by Alijagie et al. (Eur. J. Immunol. 25 (1995) 3100). Some viruses such as the adenoviruses, the AAV or the retroviruses seem capable of being used for this purpose to deliver a nucleic acid into a dendritic cell.
Preferred processes in the framework of the present invention are the sensitization methods using membrane vesicles (of the exosome type), antigenic peptides, vectors, RNAs or acidic peptide eluates of tumors (APE). The use of membrane vesicles as well as xe2x80x9cpeptide pulsingxe2x80x9d and the APE method are illustrated in the examples and are quite particularly preferred.
When the populations of dendritic cells have been obtained and optionally sensitized to one or more antigens, the dexosomes can be prepared.
This preparation comprises an optional first step of treatment of the cells, followed by a second step of isolation of the dexosomes.
The first treatment step of the cells results from the demonstration by the inventors that the production of dexosomes by the dendritic cells is a regulated phenomenon. Thus, in the absence of treatment, the quantites of dexosomes produced are relatively low. In particular, when a population of mature dendritic cells not stimulated beforehand is used, the production of dexosomes is practically undetectable. The inventors have thus shown that the production of dexosomes was essentially dependent on the type of dendritic cells and the implementation of a treatment of these cells. These preliminary elements are what make it possible to obtain dexosomes having useful properties in quantities significant for industrial use. A treatment of the dendritic cells is thus advantageously performed so as to stimulate the production of dexosomes by these cells. This stimulating treatment may be performed either by the culture of the cells in the presence of certain cytokines, or by irradiation of the cells, or by lowering the pH of the culture, or by combining these different types of treatment.
In the first embodiment the dendritic cells are incubated in the presence of a cytokine selected preferably from gamma interferon (IFNxcex3), interleukin-10 (IL-10) and interleukin-12 (IL-12), and preferably gamma interferon and IL-10. As illustrated in the examples, these cytokines seem to exert a quite pronounced stimulating effect on the production of dexosomes (factor 3 to 5). Furthermore, surprisingly, no stimulating effect was observed in the presence of the following cytokines: IL-1xcex2, IL-2, IL-4, IL-6 and IL-15, and an inhibitory effect has even been observed in the presence of lipopolysaccharide (LPS) or TNFxcex1, which are however described as stimulating the maturation of the dendritic cells. Hence these results show (i) the regulated character of the production of dexosomes and (ii) the specific effect of certain cytokines on this production. In addition, these results illustrate the surprising value of using immature dendritic cells, and the use, in the stimulation step, of cytokines inducing an immature state of the cells, such as IL-10 in particular. In this embodiment, the cytokines are used at doses adjustable by the specialist skilled in the art as a function of (i) the cytokine, (ii) the cell population and (iii) possible performance of other treatments. It is understood that the cytokines are preferably used at subtoxic doses. The doses of interleukin are usually comprised between 1 and 100 ng/ml, and preferably between 1 and 50 ng/ml. The interferon may be used at doses comprised between 1 and 500 IU/mil, and preferably between 5 and 200 IU/ml.
In the second embodiment, the dendritic cells are subjected to irradiation. The results presented in the examples indeed show that irradiation of the cells also makes it possible to increase the production levels of dexosomes. Irradiation is usually performed at between 1000 and 5000 rads, and preferably between 2000 and 4000 rads, and most favourably at approximately 3000 rads.
The second step consists of the isolation of the dexosomes. The objective of this step is to separate the dexosomes from the dendritic cells and/or the culture medium. This step makes it possible in particular to obtain a composition enriched in dexosomes and essentially free of intact cells. Preferably, this step leads to a composition comprising at least 70% and preferably at least 85% of dexosomes.
The isolation of the dexosomes may be carried out according to different separation procedures for biological materials. As described previously for the texosomes of tumor cells, these processes may be based on the differences of size, mass, charge or density of the dexosomes.
Thus, the dexosomes may be isolated by centrifugation of the culture medium or the culture supernatant or membrane fractions or lysates of dendritic cells. The isolation may be done for example by a differential centrifugation and/or density gradient cenntrifugation, followed by recovery of the fraction(s) containing said dexosomes. This type of methodology is based on the separation, by means of successive centrifugations, of the membrane vesicles on the one hand and cells, cellular debris, internal vesicles, etc., on the other. In this particular embodiment, the fraction containing the dexosomes is usually that obtained after ultracentrifugation at 100,000 g. This method is illustrated in particular in Examples 1 and 8.
The isolation step of the dexosomes may also be performed by chromatography, electrophoresis and/or nanofiltration.
A liquid phase and/or density gradient electrophoresis may be advantageously performed. The liquid phase electrophoresis, which leads to the separation of biological materials according to their charge, is quite advantageous. Example 11 below indeed shows this procedure may be profitably used for the isolation of exsomes in good yields. This procedure is, moreover, particularly advantageous on an industrial scale.
Purification may also be performed by chromatography. Mention may be made in particular of ion exchange chromatography, gel permeation (or exclusion) chromatogrphy or hydrophobic chromatography. In view of the lipid nature of the dexosomes, ion exchange chromatography is particularly useful. Nanofiltration may be performed according to known procedures on cell supernatants.
Recourse to chromatographic and/or electrophoretic procedures and/or nanofiltration constitutes another important feature of the present invention since it allows, compared to current technologies, the production of improved quality in quantities suitable for industrial use (in particular pharmacological).
In this respect, the invention also relates to a method of preparation of membrane vesicles comprising at least a separation step by electrophoresis, chromatography or nanofiltration. This process is more particularly suited to the preparation of membrane vesicles of the exosome type, such as texosomes or dexosomes. In this process, the separation step by electrophoresis or chromatography may be performed directly on a culture supernatant, a cell lysate or a pre-purified preparation. The electrophoresis is more preferably a liquid phase electrophoresis.
The dexosomes exhibit remarkable properties which are illustrated in the examples. Thus, the dexosomes stimulate the proliferation of cytotoxic T lymphocytes in vitro. Furthermore, the dexosomes are capable of blocking tumor growth in vivo. These vesicles are thus capable of presenting antigens of interest very efficiently in combination with class I and class II molecules of the MHC. The dexosomes hence have very many uses in the fields of cancer and parasitic or infectious diseases, for example. In addition, at high doses (likely to induce tolerance), the dexosomes may also be used in the treatment of diseases such as allergy, asthma or autoimmune diseases. In addition, the xe2x80x9cnaivexe2x80x9d dexosomes may also be used as adjuvant to stimulate and/or modulate an immune response.
The object of the invention is also the use:
of texosomes such as defined above, or
antigen-presenting cells such as defined above, or
dexosomes such as defined above,
for the stimulation and, optionally, the amplification in vitro of T lymphocytes specific for antigens contained in above-mentioned texosomes, antigen-presenting cells or dexosomesxe2x80x94or of B lymphocytes, and in particular for the stimulation and amplification in vitro of T lymphocytes.
The invention also relates to the use of texosomes such as defined above, or of antigen-presenting cells such as defined above or of dexosomes such as defined above for the ex vivo selection of a repertoire of T lymphocytes capable of recognizing specific antigens contained in above-mentioned texosomes, antigen-presenting cells or dexosomes.
The object of the invention is also a medicine containing as active substance at least one texosome such as defined above, one antigen-presenting cell such as defined above and/or a dexosome such as defined above, in combination with a pharmaceutically acceptable vehicle.
Advantageously, the invention relates to a medicine such as defined above for use in the treatment of cancers, parasitic or infectious diseases.
More preferably, the medicine contains texosomes or dexosomes such as defined above.
According to another embodiment, the invention relates to a medicine such as defined above for use in the treatment of the diseases of the allergy or asthma type or autoimmune disease.
As appropriate formulation, the texosomes or dexosomes may be contained in physiological serum in an ampoule or any other appropriate means (syringe, pouch, etc). They may be prepared immediately prior to use or stored, for example frozen at xe2x88x9280xc2x0 C. The solutions used may be composed of saline solutions, optionally supplemented with stabilizing agents and/or adjuvants. The stabilizing agents may be in particular proteins or molecules of high molecular weight. Mention may be made more particularly of proteins such as human serum albumin or molecules such as dextran or poloxamer, for example.
The compositions of the invention may also contain or be used in combination with one or more adjuvants. The adjuvant may be more particularly any immunostimulating pharmacological agent such as for example a cytokine (in particular interleukin-12). Such agents are classically used in the clinical protocols or in vaccinating compositions. Moreover, the adjuvant according to the invention may also be an agent capable of stimulating the production of dendritic cells in vivo. As an example, mention may be made of compound Flt3. The combined use of this type of agent makes it possible to increase the number of dendritic cells and thus to improve potentially the efficacy of the compositions of the invention.
Another object of the invention thus relates to a combination of texosomes and/or dexosomes and an adjuvant for the purpose of simultaneous use, separate use or use of each at intervals.
An appropriate mode of administration of the medicines of the invention consists of injections, and in particular intradermal or subcutaneous injections. This mode of administration is particularly suitable when the active substance of the medicine is constituted of dendritic cells loaded with texosomes or dexosomes.
The appropriate dosages are 0.01 to 10, and in particular 0.10 to 5 and even more particularly 0.15 to 2 xcexcg/kg of body weight, and 10 xcexcg for the intradermal reaction tests.
The medicines of the invention may also be used at 100 xcexcg for prophylactic vaccination treatments.
The objectives designed to be attained by the use of the medicines of the invention are:
delayed hypersensitivity (tests in cancer patients), or
prophylactic therapy, or
use in the framework of the detection of the frequency of specific cytotoxic lymphocytic precursors or secretors of interferon by the limiting dilution process.
The objective is to use the autologous or allogeneic dendritic cells preincubated with the texosomes of the invention as targets of peripheral lymphocytes of subjects bearing tumors before, during and after anti-tumor treatment (standard treatment or specific active immunization).
The invention also relates to the use of a texosome such as defined above or antigen-presenting cell such as defined above or of a dexosome such as defined above for the preparation of a medicine designed for the treatment of tumors, in particular solid, ascitiic and hematopoietic tumors.
As solid tumors, mention may be made of: cancer of the kidney, breast, colon, lung, stomach, liver, melanomas, sarcomas, etc . . .
As hematopoietic tumors, mention may be made of: leukemias, Hodgkin and non-Hodgkin malignant lymphomas.
As indicated previously, the compositions of the invention, in particular the compositions containing dexosomes, can also be used for the treatment of parasitic or infectious diseases. For this type of use, the dexosomes are loaded with antigens or peptides of the parasite or the infectious agent (virus).
The invention also relates to the use of a texosome such as defined above or of a dexosome as defined above in the framework of a delayed hypersensitivity test of the cancer or also as diagnostic tool to investigate the frequency of specific cytotoxic CTL precursors.
The invention also relates to the use of a texosome or a fraction or a constitutive constituent of a texosome such as defined above or of a dexosome such as defined above for the transfer of biological material into a cell in vitro or in vivo.
The invention also relates to the creation of texosome libraries derived from tumor cells of a common or different histological type.
These latter are composed of mixtures of texosomes made from tumor cell lines for a given type of cancer. These texosome libraries may enable antigen-presenting cells, in particular dendritic cells, to be sensitized against all the tumors of this type.
The invention also relates to mixtures of texosomes or dexosomes.
For example, mention may be made of mixtures of texosomes for genetically related tumors (cancer of the breast and ovary) or exhibiting known mutations p53, p16 (breast cancer, sarcoma).
Mention may also be made of mixtures of tumor texosomes with vesicles derived from immortalized cells and transfected to express co-stimulatory molecules, adhesion molecules, attractant chemokines (different from those expressed on the texosomes).
The present invention will be described in more detail with the aid of the examples which follow, which must be considered as illustrative and not limiting.